The present invention relates to the field of chemistry and biochemistry and, in particular, to the analytical methods and apparatuses for loading, unloading and analyzing samples by atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) technique.
Mass spectrometers have become one of the essential tools of the biochemistry lab. Biochemists take advantage of the capabilities of mass spectrometers to determine molecular weights of biomolecules, monitor bioreactions, detect post-translational modifications, perform protein, and oligonucleotide sequencing, and many more applications. During the past decade, one of the methods, which became most successful for the mass spectrometric analysis and investigation of large molecules is a method known as MALDI (Matrix-Assisted Laser Desorption Ionization). This method, which in application to time-of-flight (TOF) mass spectrometry (MS) is known as MALDI-TOF MS, is a relatively novel technique in which a co-precipitate of an UV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. The sample (analyte) is suspended or dissolved in a matrix (e.g., in 1000xc3x97 molar excess).
Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. Matrices are small organic compounds that are co-crystallized with the analyte. It seems that the presence of the matrix, spares the analyte from degradation, resulting in the detection of intact molecules as large as 1 million Da. The ionized biomolecules are accelerated in an electric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass to charge ratio and reach the detector at different times. In this way each molecule yields a distinct signal. The method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides and oligonucleotides, with molecular masses between 400 and 350,000 Da. It is a very sensitive method, which allows the detection of low (10xe2x88x9215 to 10xe2x88x9218 mole) quantities of sample with a mass accuracy of 0.1-0.01% or higher.
Another advantage of MALDI is that this method allows for vaporization and ionization of non-volatile biological samples from a solid-state phase directly into the gas phase.
The most important step in MALDI, is sample preparation. During this step, the matrix and analyte are mixed and the mixture is dried on a probe or as it is more common now, on a sample plate. Upon preparation, the sample plate with samples is loaded into the mass spectrometer.
A laser beam, serves as the desorption and ionization source in MALDI. The matrix plays a key role in this technique by absorbing the laser light energy and causing part of the illuminated substrate to vaporize. A rapidly expanding matrix plume carries some of the analyte into the vacuum with it and aids the sample ionization process. The matrix molecules absorb most of the incident laser energy minimizing sample damage and ion fragmentation (i.e., soft ionization). Nitrogen lasers operating at 337 nm (a wavelength that is well absorbed by most UV matrices) are the most common illumination sources because they are inexpensive and offer the ideal combination of power/wavelength/pulsewidth. However, other UV and even IR pulsed lasers have been used with properly selected matrices.
Once the sample molecules are vaporized and ionized, they are transferred, e.g., into a time-of-flight mass spectrometer (TOF-MS) where they are separated from the matrix ions, and individually detected, based on their mass-to-charge (m/z) ratios and analyzed. High transmission and sensitivity, along with theoretically unlimited mass range are among the inherent advantages of TOF instruments. Detection of the ions at the end of the tube is based on their flight time, which is proportional to the square root of their m/z.
Roughly, the MALDI system can be divided into two groups: VP-MALDI for matrix-assisted laser desorption at vacuum pressure and AP-MALDI for matrix-assisted laser desorption at atmospheric pressure. A characteristic feature of vacuum-pressure ionization sources is that sample ionization occurs inside a mass spectrometer housing under vacuum conditions. In contrast to vacuum ionization, any atmospheric pressure ionization takes place outside a mass spectrometer instrument. It should be noted that different instrument types are used in both cases. However, for sampling atmospheric pressure ions any mass spectrometer must be equipped with Atmospheric Pressure Interface (API) to transfer ions from an external region of atmospheric pressure to a mass analyzer under high vacuum.
Examples of both these systems are given below for more detailed familiarization with the MALDI technique.
U.S. Pat. No. 5,288,644 issued in Feb. 22, 1994 to Ronald C. Beavis, et al. discloses an apparatus and method for the sequencing of genome. The apparatus comprises an automated DNA sampler, which adds a matrix solution from a container to separated DNA samples. A large number of DNA fragment samples, for example 120 samples, may be loaded into a sample tray. The matrix solution may be added automatically to each sample using procedures available on the aforementioned autosampler, and the samples may then be spotted sequentially as sample spots on an appropriate surface, such as the planar surface of the disk rotated by a stepper motor. Sample spot identification is entered into the data storage and computing system, which controls both the autosampler and the mass spectrometer. The location of each spot relative to a reference mark is recorded in the computer. Sample preparation and loading onto the solid surface is done off-line from the mass spectrometer, and multiple stations may be employed for each mass spectrometer, if the time required for sample preparation is longer than the measurement time.
Once the samples in suitable matrix are deposited on the disk, the disk may be inserted into the ion source of a mass spectrometer through the vacuum lock. Any gas introduced in this procedure must be removed prior to measuring the mass spectrum. Loading and pump down of the spectrometer typically requires two to three minutes, and the total time for measurement of each sample to obtain a spectrum is typically one minute or less.
Thus, the above-described VP-MALDI technique requires that the sample plate carrier be loaded into the mass spectrometer through a vacuum lock and even though manipulations with the sample support are carried out automatically and coordinated by the computer, all sampler operations for loading the samples into the mass spectrometer are performed in vacuum. Such a system requires the use of complicated vacuum seals and special drive, transportation, and actuation mechanisms, which have to be vacuum-proof. Furthermore, the sample loading system of U.S. Pat. No. 5,288,644 has a relatively low throughput rate. As the authors of the above invention states, the system is limited to about 50 complete DNA spectra per hour. Furthermore, the system is expensive, as it requires the use of vacuum-proof sample plate carrier handling mechanisms.
U.S. Reissued Patent RE 37,485 filed by Marvin L. Vestal and published on Dec. 25, 2001 describes another mass spectrometer system and method for vacuum-pressure matrix-assisted laser desorption measurements. The system is equipped with a sample plate transport mechanism for automatically inputting and outputting each of the sample plates into and from the sample-receiving chamber of the mass spectrometer.
A sample support or plate used in the aforementioned system comprises a thin, substantially square plate of stainless steel or other suitable electrically conducting material approximately 1.5 mm thick and 50 mm wide. The plate may contain precisely located holes to allow the position and orientation of the plate to be accurately determined relative to a moveable stage, which is required both in the sample loading step and in the ion source of the mass spectrometer. The sample plate also contains a plurality of precisely determinable sample positions on the upper sample-receiving surface of the plate. The sample plate may thus contain 100 sample positions each identified by a sample spot, which is about 2.5 mm in diameter in a precisely known location on the plate, with each sample support being suitable for accepting a few microliters of sample solution.
The sample plate is rigidly attached to a ferromagnetic material handle, which is used to engage an electromagnetic device for the purpose of transporting the sample plate between component systems. The sample plate has two or more precisely located holes which locate the sample plate carrier when installed in the sample receiving stage in the ion source of the mass spectrometer and in the sample transport trays.
Processing and preparing samples may be different, depending on the application, the types of samples to be tested, and the degree to which the samples are prepared and purified prior to being input to the analysis system.
The sample processing components include an autosampler, valves for controllably adding an appropriate solution of matrix from containers to each sample, and a pump or another flow system for transferring liquid samples from a selected sample to a known sample position on the sample plate. The sample plate is precisely located on a carrier mounted on a computer-controlled x-y table. Each sample position may be computer recorded at the time the sample aliquot is transferred to the plate. The autosampler may be similar to autosamplers used with capillary electrophoresis. The above-described sample input and preparation operations are carried out under atmospheric pressure prior to loading to the vacuum chamber of the mass spectrometer.
When each sample location on a plate has been loaded with a sample, the samples are allowed to dry before the plate is transferred into the vacuum chamber of the mass spectrometer. In the simplest case, the plates may be transferred from the sample loading system to a rack or cassette where they are allowed to dry in laboratory air, and preferably in a sealed chamber equipped with a computer-controlled door which allows the samples to be dried in an environment in which the pressure, temperature, and composition of the surrounding atmosphere is controlled. In the fully automated mode, each of the loaded and dried sample plates may be transferred from the sample plate storage chamber or cassette to an adjacent mass spectrometer.
The manual step involved in loading the sample plates may be eliminated by adding to the vacuum lock chamber of a mass spectrometer a sample storage region. This provision, when coupled with on-line sample loading, allows the system to be operated in a fully automatic, unattended mode. In this configuration, an input door is located between the vacuum lock chamber and the storage chamber. An air cylinder transporter equipped with electromagnets is provided for transporting sample plates from the transport tray within the storage chamber to the vacuum lock chamber. The tray contains multiple shelves and corresponding slots each for storing a sample plate. A cassette transport device mechanism including a lead screw driven by a stepper motor is provided to allow any selected one of these slots and a corresponding plate in the cassette to be brought into line with transporter.
The above-described loading system allows the sample plates to be loaded into the storage region of the vacuum lock chamber, while another sample plate is being analyzed in the ion source chamber of a mass spectrometer. In fully automatic operation, whenever a new sample plate may be loaded, the storage chamber is evacuated, the input door between the storage chamber and the vacuum lock chamber is opened, and the new sample plate is automatically moved by transporter to a sample transport tray provided in the vacuum lock chamber. The input door is then closed and the vacuum lock chamber remains evacuated. The plate positioned by the sample transport tray is moved within chamber by an air cylinder transport mechanism.
When analysis of the samples on one plate within the ion source is completed, the plate is ejected and placed in a vacant slot in the sample storage cassette. This cassette is then moved by a stepper motor and a lead screw to bring a new sample plate in the transport tray.
Thus, as has been shown above, the VP-MALDI system of U.S. RE 37,485 is characterized by the use of a plurality of sample supports which are transferable between the sample preparation station and the mass spectrometer with the use of a x-y transport mechanism so that when one of the sample supports is located inside the vacuum chamber of the mass spectrometer, other sample supports are loaded with the samples and prepared for the analysis. However, the transfer from the preparation station to the vacuum chamber and in the opposite direction is always carried out through the vacuum lock. This intermediate transfer operations delays the throughput of the system as a whole since before loading a sample plate into the storage chamber for subsequent analysis by the mass spectrometer, the sample loading doors of the vacuum lock should be closed, the pumpout valve should be connected to the mechanical vacuum pump, the latter should be open, the sample storage chamber should be evacuated to a predetermined acceptable vacuum level (e.g., 20 millitorr), input and output doors should be opened and allow sample plates to be transported between the sample storage chamber and the ion source chamber of the mass spectrometer without significantly degrading the vacuum of the mass spectrometer. Execution of these sequential operations requires time and therefore, in spite of a provision of fully automated loading/unloading mechanisms, the VP-MALDI system has some limitations with regard to the system throughput. Furthermore, a provision of the sample storage chamber with an appropriate valve system for evacuation of this chamber makes the structure of the system more complicated and expensive.
Another disadvantage of the sample plate delivery system of the aforementioned reissued patent is that two different mechanisms are required for picking up and delivery of the sample plates to the location from where the samples are introduced into the mass spectrometer (in the above case, to the vacuum lock) and for retaining the sample plate in the aforementioned location.
A serious disadvantage of the aforementioned system is that for picking up sample plates the handling mechanisms come in direct contact with each sample plate, and this increases a chance of contamination of the samples and of the sample plates themselves.
Another serious disadvantage of the automatic loading/unloading systems for VP-MALDI is that the mechanisms for operation in vacuum must be provided with reliable vacuum seals and therefore such mechanisms are complicated in structure and very expensive to manufacture. They also are expensive and complicated in maintenance.
In order to eliminated disadvantages of the vacuum-pressure MALDI, an atmospheric-pressure MALDI (hereinafter referred to as AP-MALDI) system was developed. At the present time, the AP-MALDI has become a technique that competes in sensitivity and selectivity with conventional VP-MALDI. In the AP-MALDI, the samples deposited on sample plates do not need to be delivered into the vacuum chamber, and only the plum of ions produced by laser radiation is sampled into an atmospheric pressure ionization mass spectrometer.
An example of a AP-MALDI system is the one disclosed in U.S. Pat. No. 5,965,884 issued on Oct. 12, 1999 to Victor V. Laiko, et al. This AP-MALDI apparatus comprises an ionization chamber, an interface for connecting the ionization chamber to a spectrometer, a sample plate or support with sample deposited on its target surface, a laser, and a lens for focusing a laser beam generated by the laser.
The ionization chamber is used to contain a bath gas or gas mixture, which is at atmospheric pressure or near atmospheric pressure. Dry nitrogen and dry air is normally used as the bath gas. A gas inlet is incorporated in the gas chamber, which provides the pathway for the bath gas to enter the ionization chamber. The ionization chamber also has a window for the laser beam to enter the chamber. Additional equipment can be incorporated into the ionization chamber to further control the humidity, the temperature and the pressure of the bath gas.
The interface, which is usually a part of the spectrometer, comprises an inlet orifice, through which ionized analyte particles enter the spectrometer from the ionization chamber. The inlet orifice is connected to a electric power supply to serve as an electrode.
The sample support is also connected to an electric power supply, which also serves as an electrode. The two electrodes of the inlet orifice and the sample support provide the electric field which helps move the ionized analyte from the sample support to the inlet orifice. The electric potential applied to the electrodes is adjusted to optimize the signal level measured by the spectrometer.
The sample is deposited on a target surface of the sample support, which is aligned with the inlet orifice of the interface to facilitate the ionized analyte to move to the inlet orifice.
The laser positioned outside the ionization chamber is a UV laser, a visible laser, or an IR laser. The laser beam is focused by a lens. The position of the lens is adjusted so that best measurement results are achieved by the spectrometer.
The AP-MALDI has the following advantages as compared to the VP-MALDI. The AP-MALDI takes place under atmospheric pressure conditions. This allows a more or less uniform ion cloud to form after laser illumination, because the produced ions achieve a thermal equilibrium with the surrounding bath gas molecules quickly through collision. As a consequence, the AP-MALDI technique produces a quasi-continuous ion source, which provides a stable ion supply to a mass spectrometer.
A more powerful laser pulse can be used in AP-MALDI because vibrationally excited analyte ions are quickly thermalized (stabilized) with the surrounding bath gas molecules before they dissociate into fragments. Furthermore, a larger laser spot is used to illuminate the sample, which allows an easier alignment procedure in comparison with the vacuum MALDI technique. As a consequence, substantial amount of ions, as much as a few picomoles, are generated in AP-MALDI to compensate for the loss due to API.
AP-MALDI has an ion source, which is external with respect to the spectrometer instrument. Thus any mass spectrometer equipped with Atmospheric Pressure Interface (API) may be easily coupled with this ion source without undue effort. The de-coupling of ion source from the ion-focusing optics of a spectrometer ensures the same resolution level and spectra calibration procedure as for any other atmospheric pressure ionization technique. As a result, different mass analyzers, such as quadrupole ion-trap type, time-of-flight type, and Fourier transform ion cyclotrone resonance type or mass analyzers of other types may be easily coupled with AP-MALDI.
Atmospheric pressure character of AP-MALDI allows a simple sample loading procedure. Consequently, the construction of the instrument is simplified drastically. Both sample preparation and ionization processes take place under atmospheric pressure conditions.
AP-MALDI is a versatile technique. The selection of possible matrix material for AP-MALDI is not limited to solids or liquid matrixes with very low vapor pressures. The most important feature of the AP-MALDI as compared to the VP-MALDI is that matrixes of volatile liquids may be used only under atmospheric pressure conditions without the use of any special procedures and devices.
However, a disadvantage of the atmospheric-pressure system of U.S. Pat. No. 5,965,884 is that it is associated with manual loading/unloading procedures of the sample supports.
U.S. Pat. No. 6,541,768 issued on Apr. 1, 2003 to Bruce A. Andrien, Jr., et al. discloses a multiple sample introduction system for API (atmospheric pressure ionization) mass spectrometry. In this system, the electrospray ion source is interfaced to a mass spectrometer, which is configured in vacuum chamber. Individual electrospray (ES) probe assemblies can be configured in the electrospray ion source atmospheric pressure chamber, where solution is sprayed from individual probe tips. In one of the embodiments, the electrospray source is configured with an ES probe assembly comprised of six ES tips with individual liquid supply lines. A position adjuster can be used to move ES probe assembly such that any ES tip can be located near the ES source centerline. With this device several sample solutions can be rapidly analyzed with little or no cross contamination which can occur when multiple samples are delivered to the ES source sequentially through the same ES probe tip. Although the system described above is intended for introduction of the samples to the vacuum chamber of the mass spectrometer from the atmospheric pressure chamber, the API system described above is not designed for automatically inputting and outputting each of the sample supports into and from the sample receiving chamber of the mass spectrometer. The system is rather intended for individual introduction of sample supports one-by-one in a slow sequence and therefore does not need means for automating loading or unloading of ES tips. The ampoule-like construction of the ES-tips itself is not suitable for quick introduction of the sample into the mass spectrometer, as compared to the use of a multiple-cell sample plates.
Agilent Technologies Inc. introduced an AP-MALDI, co-developed by Masstech, Inc., by providing a sample handling interface and a laser that is capable of handling and analyzing a sample plate with multiple samples. This interface with little or no modification can be attached to various atmospheric pressure ionization mass spectrometers such as an ion-trap type, and a time-of-flight mass spectrometers. In this apparatus, a substrate plate with deposited samples is manually loaded on a target flange that is equipped with positioning x-y stage. The x-y stage is used to move the sample plate with respect to the mass spectrometer sampling orifice to analyze samples deposited on the plate. The main disadvantage of the apparatus is the inconvenience of manual loading for sample plates. The autosamples, which are designed for the conventional vacuum MALDI, are too complicated and inefficient for AP-MALDI since they are designed for delivering of the sample plates into the vacuum. Also robotic systems used in stamping or welding industries with a flexible arm can be used to load the sample plates, into a commercial apparatus produced by Masstech, Inc., however these systems are complex and not designed to work directly with plates that have biological sample deposited on one side of the plate. For example, for picking up plates several commercial robotic systems use vacuum grips that can destroy or contaminate the deposited sample.
Thus, it has been shown that in all known systems of sample analysis equipped with automatic or semi-automatic sample plate loading and transportation mechanisms the grippers of these mechanisms come in physical contact directly with the sample plate that carriers the samples. This condition creates possibility for contamination of the samples and sample plates.
It is an object of the present invention to provide an apparatus for automated sample analysis by MALDI mass spectrometry, wherein the number of contacts between the sample plate and a sample plate handling mechanism is reduced due to the use of an intermediate sample plate carrier for carrying and handling the sample plates prior to loading them to the working station of the mass spectrometer.
It is another object to provide a moveable and disconnectable sample-plate handling flange, which is docked to the orifice inlet port of the mass spectrometer during loading of the samples to the mass spectrometer and is provided with an additional function of transfer of the sample plates from the storage cassette to the mass spectrometer. It is another object of the invention to provide the apparatus of the aforementioned type with sample plates carriers that are used as an intermediate sample plate carriers for carrying the plates to a pick-up station and that circulates between the aforementioned station and a sample plate carrier storage device.
The apparatus of the invention consists of two module, one of which carries sample-plate handling flange, which is docked to the mass spectrometer in a working position for loading ionized samples from the atmospheric pressure environment to the to the mass spectrometer, while the other one is used for picking up the sample plate carriers with preliminarily prepared sample plates from the storage cassette and for transferring the aforementioned carriers to a stand-by position for disconnection of the sample plates from the carriers and for deliver them to the mass spectrometer. The sample-plate handling flange is disconnectable from the mass spectrometer and moveable between the aforementioned working position and the stand-by position and is provided with means for taking the stand-by sample plate from the plate carrier and for holding the plate with samples during delivery of the ionized samples to the mass spectrometry. The sample-plate handling flange is also provided with means for shifting the sample plate inside the sample-plate handling flange in the X-Y coordinate system for arranging a selected sample cell coaxially with the center of the ion-sampling orifice. The first module provides movements in the Z-axis and X-axis directions, and the second module provides movements in the Z-axis and Y-axis direction. As the mechanisms of the aforementioned modules operate under atmospheric pressure, they do not need the use of any special and expensive sealing devices required for use of similar mechanisms in vacuum. The system is fully automated and movements of all mechanisms and drives are controlled by a data preliminarily inputted to a central processing unit provided in the control system of the apparatus.